P1564: FAMILIAL ERYTHROCYTOSES IN THE CZECH REPUBLIC - GENETIC CHARACTERIZATION AND HEPCIDIN REGULATION
Authors: L. Sochorcová, K. Hlusickova Kapralova, O. Jahoda, J. Manakova, E. Kriegova, B. Kralova, J. Fialova Kucerova, M. Divoka, D. Prochazkova, D. Pospisilova, V. Divoky, M. Horvathova
Journal: HemaSphere (2022)

Background: Absolute erythrocytosis is a condition characterized by elevated erythrocyte mass. Familial erythrocytoses represent a group of rare inherited red blood cell disorders caused by mutations in genes involved in erythropoietin receptor (EPOR) signal transduction, hypoxia signaling pathway, or hemoglobin-oxygen affinity regulation. Nevertheless, up to 70% of patients have unknown etiology. Similarly, the coordination of erythropoietic activity with iron homeostasis in different subtypes of familial erythrocytoses needs to be clarified. Aims: We performed molecular-genetic analysis of 13 pediatric patients with inherited erythrocytosis from the Czech Republic and assessed co-regulation of erythropoiesis and iron metabolism, including evaluation of hepcidin and its negative erythroid regulator erythroferrone (ERFE). Methods: Genomic DNA was used for Sanger sequencing, next generation sequencing (NGS), or whole exome sequencing (WES). The sensitivity of erythroid progenitors to erythropoietin (EPO) was determined in vitro. Serum EPO levels and iron status parameters were measured; hepcidin and ERFE levels were determined with commercial ELISA kits. Results: Using targeted Sanger sequencing we found a causative mutation in 5/13 patients from the cohort. Three unrelated patients with low EPO levels and hypersensitivity of erythroid progenitors to EPO harbored mutations in EPOR. A known heterozygous c.5967insT EPOR mutation was identified in two of them; the third patient carried a novel heterozygous c.11092delTG EPOR mutation. Both these mutations lead to a truncation of EPOR. A previously reported heterozygous p.G537R mutation in hypoxia-inducible factor alpha (HIF2A), encoded by EPAS1, was detected in two unrelated patients with normal EPO levels and EPO-hypersensitive erythroid progenitors. Targeted NGS of erythrocytosis-associated genes was applied in the other 6/13 patients. The analysis was negative in all but one patient in whom a rare germline JAK2 variant (p.N1108S) was identified, known from triple-negative myelofibrosis patients and presumably predisposing to acute myeloid leukemia. The remaining 2/13 patients, siblings with plethora, splenomegaly, normal sensitivity of erythroid progenitors to EPO, and elevated/normal EPO, were subjected to WES. A known mutation in a cryptic exon of VHL gene (c.340 + 770T>C) was found in a homozygous state in both of them; their parents were heterozygous asymptomatic carriers of this mutation. We then focus on the assessment of iron metabolism; 6 patients from the cohort (2 EPOR-mutant, 2 HIF2A-mutant, and 2 VHL-mutant) were available for hepcidin and ERFE measurements. All EPOR-mutant and HIF2A-mutant patients had normal serum iron and ferritin levels, and reduced/subnormal transferrin saturation; both VHL-mutant patients were iron deficient. Slightly elevated ERFE and low/undetectable hepcidin levels were observed in HIF2A- and VHL-mutant patients. On the other hand, EPOR-mutant patients had normal hepcidin and ERFE levels below the detection threshold, suggesting that ERFE is not the main regulator of hepcidin in EPOR-mutated erythrocytosis. Summary/Conclusion: We identified disease-causing mutations in 7/13 erythrocytic patients from our cohort. Despite the use of targeted NGS, almost half of the cases remained idiopathic. In these patients, WES or WGS should be used. We also suggest that EPOR-mutant erythrocytoses differentiate from HIF2A- and VHL-mutant erythrocytoses not only in low EPO levels, but also in the regulation of systemic iron homeostasis. Grants support: NV19-07-00412; IGA_LF_2022_003.